Total RNA and oligo dT primers were incubated for 5 min at 56 C, followed by addition of 4. 90 uL of 5first strand buffer, 1. 5 uL of 0. 1 M dithio threitol, 0. 5 uL of RNAse H inhibitor, 1 uL of 10 mM dNTPs, and 0. 1 uL of SupersriptW Re verse Transcriptase III. The reaction was performed selleck screening library for 1 h at 42 C, followed by adding 80 uL of DEPC treated water Inhibitors,Modulators,Libraries and storage at ?20 C until further use. Relative quantification of mRNA ex pression levels was performed by real time RT PCR using the KAPA SYBRW FAST qPCR Kit and gene specific oligonucleo tide primers. Quantitative real time RT PCR was performed in a final volume of 20 uL and using a Rotor Gene 6000 thermal cycler. The following amplification procedure was applied initial denaturation for 15 min at 95 C, followed by 40 cycles of denaturation for 15 s at 94 C, annealing for 30 s at 56 C, and extension for 30 s at 72 C.

Three replicates Inhibitors,Modulators,Libraries were analyzed per sample. Relative gene ex pression compared with the internal control GAPDH was determined using the 2 CT method. The oligonucleotide primers for real time RT PCR are listed in Additional file 1 Table S1. Determination of IL 6 protein expression Cells grown in 96 Inhibitors,Modulators,Libraries well plates were treated for 24 h with the compounds indicated, and cell free supernatants were collected. IL 6 protein was measured using an ELISA kit from BD Biosciences. Briefly, a 96 well plate was coated with capture antibody overnight at 4 C, fol lowed by washing five times with PBS T. The wells were blocked with assay diluents for 1 h and washed five times.

Standards and collected culture supernatants were added to the appro priate wells and samples were incubated for 2 h. After rinsing five times with PBS T, each well was incubated with detection antibody for 1 h. After washing, avidin HRP was added for 30 min. After rinsing seven times, each well was incubated Inhibitors,Modulators,Libraries with substrate solution for 30 min in the dark. Reaction was stopped by adding 1 M H3PO4, and the plate was analyzed by measuring absorb ance at 450 nm and subtracting the values at 570 nm using a UV max kinetic microplate reader. IL 6 protein concentrations were calculated according to the standard curve of purified mouse IL 6. Determination of NF ��B localization by fluorescence microscopy BV 2 cells were grown on poly L lysine coated glass slides in 6 well plates.

Following treatment, the medium was removed and cells were fixed and stained using CellomicsWNF ��B Inhibitors,Modulators,Libraries p65 activation HCS kit according to the manufacturers instructions. Briefly, cells were fixed with 4% paraformaldehyde sellckchem in PBS for 15 min at 25 C, washed three times with PBS, and permeabilized with PBS con taining 0. 5% Triton X 100 for 10 min. After three washes with PBS and blocking with 1% fatty acid free bovine serum albumin for 1 h the samples were incu bated with rabbit polyclonal antibody against the p65 subunit of NF ��B for 2 h at 37 C.